Cloning and characterization of the flba gene of h. pylori, production of aflagellate strains

ABSTRACT

The present application relates to nucleotide sequences which regulate the biosynthesis of the flagella proteins  Helicobacter pylori,  to the proteins encoded by these sequences and to aflagellate bacterial strains. The invention also relates to the use of these means for detecting an infection due to  H.pylori  or for protecting against such an infection.

[0001]Helicobacter pylori (also designated an H.pylori) is aGram-negative bacterium which, to date, has been found exclusively onthe surface of the mucosa of the stomach in man.

[0002] In common with most bacteria, H.pylori is sensitive to a mediumwhich is at acid pH but, nevertheless, is able to tolerate acidity inthe presence of physiological concentrations of urea (Marshall et al.(1990) Gastroenterol. 99: 697-702). by hydrolysing the urea to formcarbon dioxide and ammonia, which are released into the microenvironmentof the bacterium, the H.pylori urease enables the bacterium to survivein the acidic environment of the stomach. Recently, studies carried outon animal models have provided data suggesting that the urease is animportant factor in the colonization of the gastric mucosa (Eaton et al.(1991) Infect. Immun. 59: 2470-2475). The urease is also suspected ofcausing injury, either directly or indirectly, to the gastric mucosa.

[0003] Currently, Helicobacter pylori (H.pylori) is recognized as beingthe etiological agent of antral gastrites, and appears to be one of thecofactors required for the development of ulcers. Furthermore, itappears that the development of gastric carcinomas may be associatedwith the presence of H. pylori.

[0004] In order to develop novel sensitive and specific means fordetecting in-vitro infections due to bacteria of the Helicobacter pylorispecies, the inventors have been taking an interest in the system forregulating the mobility of these bacteria.

[0005] With this aim in view, they have been interested in differentmodifications of the H.pylori strains, modifications which did notaffect the recognition of these bacteria by sera from infected patientsbut which nevertheless rendered it possible to avoid obtaining reactionsof the “false positive” type, in particular with bacteria of theCampylobacter family, for example Campylobacter jejuni.

[0006] Furthermore, the inventors observed that it was possible, if needbe, for the modified bacteria which were obtained to be employed inconstructing immunogenic compositions or compositions used forvaccination. In this respect, the invention proposes, in particular,live attenuated bacterial strains.

[0007] In a first step, the inventors identified and isolated the geneflbA which is involved in the regulation of the biosynthesis of theflagella of H.pylori and, as a consequence, in the regulation of themobility of the bacterium. The biosynthesis of the flagella comprisessynthesizing flagellins A and B and synthesizing the sheath. The flbAgene regulates both the synthesis of flagellins A and B and thesynthesis of the sheath which contains these flagellins. The inventorsestablished that the flbA gene was also important in that it regulatedthe biosynthesis of the anchoring protein of the bacterium, also termedthe “hook”.

[0008] The invention therefore relates to a nucleotide sequence from theflbA gene regulating the biosynthesis of the proteins of theHelicobactor pylori flagella, characterized in that it is able tohybridize, under conditions of high stringency, with a probecorresponding to a nucleotide fragment from H.pylori which has beenamplified using two oligonucleotides having the following sequences:

[0009] OLFlbA-1: ATGCCTCGAGGTCGAAAAGCAAGATG

[0010] OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTC, or able to hybridize,under conditions of high stringency, with these oligonucleotides.

[0011] Such a sequence can be obtained by the steps of:

[0012] screening a genomic library containing the chromosol DNA of anH.pylori strain with a probe corresponding to a nucleotide fragment fromH.pylori which has been amplified using two oligonucleotides having thefollowing sequences:

[0013] OLFlbA-1: ATGCCTCGAGGTCGAAAAGCAAGATG

[0014] OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTC, or able to hybridize,under conditions of high stringency, with these oligonucleotides,

[0015] recovering the DNA sequences which hybridize with the said probe.

[0016] subcloning the DNA sequences which have been obtained in anappropriate vector of the plasmid type and selecting those modifiedvectors which hybridize, under conditions of high stringency, with theprobe corresponding to the DNA fragment from H.pylori which has beenamplified using oligonucleotides OLFlbA-1 and OLFlbA-2.

[0017] sequencing the DNA fragments contained in the plasmid vectorswhich hybridize with the abovementioned probe and determining the openreading frame contained in these fragments.

[0018] Advantageously, those DNA fragments will be used to reconstitutethe coding sequence of the FlbA gene, corresponding to an open readingframe comprising approximately 2196 nucleotides.

[0019] The genomic library containing the chromosomal DNA of H.pylorican be obtained from an H.pylori strain. A cosmid library may also beprepared from the chromosomal DNA of H.pylori.

[0020] An example of a strain which can be used for constructing thislibrary is the strain N6, which was deposited in the NCIMB on Jun. 26,1992 under No. NCIMB40512.

[0021] The two oligonucleotide primers which are used for preparing theprobe which is intended for hybridizing the sought-after DNA which ispresent in the H.pylori DNA library are selected from the conservedregions of the various proteins of the LcrD/FlbF family.

[0022] The two oligonucleotide primers, OLFlbA-1 and OLFlbA-2, enabled afragment to be amplified which was usable as a probe and which was of130 base pairs, having the following sequence:

[0023] ATG CCA GGA AAG CAA ATG GCG ATT GAT GCG GAT TTA AAT TCA

[0024] GGA CTT ATT GAT GAT AAG GAA GCT AAA AAA CGG CGC GCC GCT

[0025] CTA AGC CAA GAA GCG GAT TTT TAT GGT GCG ATG GAT GGC GCG

[0026] TCT AAA TTT

[0027] The conditions of high stringency referred to above are thefollowing: the hybridization is carried out at 42° C. in the presence of50% formamide in a 2×SSC buffer containing 0.1% SDS (1×SSC correspondsto 0.15 M NaCl plus 15 mM sodium citrate−pH 7.0). The washings arecarried out at 68° C., for example twice during a period of one hour,using 2×SSC plus 0.1% SDS.

[0028] A nucleotide sequence which is particularly interesting inaccordance with the invention is the sequence of the flbA genecorresponding to the sequence of nucleotides depicted in FIG. 2, or to anucleotide sequence which hybridizes, under conditions of highstringency, with the abovementioned sequence.

[0029] According to another embodiment of the invention, the nucleotidesequence which is the subject-matter of the present application ischaracterized in that it encodes a protein having the amino acidsequence depicted in FIG. 2 or an amino acid sequence possessing thesame regulatory properties, with regard to the biosynthesis of theflagellar proteins of H.pylori, as the abovementioned sequence.

[0030] The invention also relates to a nucleotide sequence whichcorresponds to the previous definitions and which is modified bydeletion, substitution or insertion of bases or of a fragment of anucleotide sequence, such that:

[0031] either the flbA gene is no longer expressed in a host cell,

[0032] or the expression of the flbA gene in a host cell does not enablethe A and B flagellins or the sheath which contains them to bebiosynthesized and, if this is the case, does not enable the H.pylorianchoring protein or the hook, to be synthesized.

[0033] The modification to which the nucleotide sequence of theinvention is subjected should be such that it is irreversible and, inparticular, that it remains irreversible when this sequence isrecombined with the flbA gene which is present in a bacterium which istransformed with a nucleotide sequence which is modified in this manner.This recombination is, for example, of the “double crossing over” type.Preferably, the modification of the nucleotide sequence should notinvolve any substantial modification—after replacement, by this modifiedsequence, of the corresponding fragment of the normal flbA gene in agiven H.pylori strain—of the functions of the neighbouring genes.

[0034] Also included within the scope of the invention are nucleotidesequences which constitute a fragment of the flbA gene meeting the abovecriteria. As examples, fragments which are the subject-matter of theinvention consist of at least 6 nucleotide sequences, preferably atleast 50, if not at least 100 nucleotides.

[0035] Such fragments are, for example, selected either on account oftheir specific flbA gene character or because they belong to conservedregions of several genes encoding proteins of the LcrD/FlbP family.

[0036] According to another embodiment, the invention is also directedtowards the fragments of the flbA gene which are delimited by therestriction sites which are present in the gene. Some of these sites aredefined, by way of example, in FIG. 1B.

[0037] Another fragment according to the invention is a fragment of atleast 1000 bp which is derived from any region of the flbA gene andwhich preferably includes a restriction site or is capable ofaccommodating a restriction site.

[0038] Other nucleotide sequences of the invention are, for example,recombinant nucleic acids which comprise a nucleotide sequence such asthose which have bee described above, itself modified by the insertionof a cassette containing a marker, for example a gene for resistance toan antibiotic or a gene for resistance to a heavy metal such asdescribed in Application FR 9406202, which was filed on May 20, 1994.

[0039] Thus: a cassette for resistance to kanamycin can be inserted.Various techniques can be used in this context and reference is made, inparticular, to the paper of Labigne A. et al. (J. of Bacteriology, Vol.170, 1988, p 1704-1708) and the paper of Labigne A. et al. (Res.Microbiol 1992, 143, 15-26).

[0040] The invention also relates to specific oligonucleotides from apreviously defined nucleotide sequence, which oligonucleotides arecharacterized in that they possess one of the following sequences:

[0041] OLFlbA-1: ATGCCTCGAGGTCGAAAAGCAAGATG

[0042] OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTC

[0043] OLFlbA-7: CGGGATCCGTGGTTACTAATGGTTCTAC

[0044] OLFlbA-8: CGGGATCCTCATGGCCTCTTCAGAGACC

[0045] According to another embodiment, the invention relates to anamino acid sequence from the FlbA protein of H.pylori, which sequence ischaracterized in that it is encoded by a nucleotide sequence such aspreviously defined.

[0046] A specific amino acid sequence from the flbA protein of H.pylori,is depicted in FIG. 2.

[0047] Thus, within the scope of the invention, the flbA gene and theprotein expressed by this gene can be of interest, in particular foremployment in immunogenic compositions or compositions used forvaccination.

[0048] The invention is also directed towards bacterial strains ofHelicobacter pylori which possess an aflagellate phenotype, whichphenotype results from the mutation, by substitution, addition and/ordeletion of bases or of a nucleotide fragment, of the above-definednucleotide sequences of the flbA gene involved in the regulation of thebiosynthesis of the flagellar proteins of H.pylori.

[0049] This modification of the flbA gene makes it possible to obtain astrain of the aflagellate type, that in which no longer expresses theFlaA and FlaB proteins and which preferably no longer expresses theproteins of the sheath.

[0050] According to one embodiment of this bacterial strain, the strainwhich is obtained additionally lacks the hook protein of H.pylori.

[0051] Preferably, a bacterial strain which meets the abovementionedcriteria is characterized in that it is obtained from the strain NG,which was deposited in the NCIMB on Jun. 26, 1992 under number NCIMB40512.

[0052] By way of example, the invention relates to a recombinantaflagellate strain of H.pylori which is designated N6flbA- and wasdeposited in the NCIMB on Jun. 30, 1995 under the No. NCIMB 40747.

[0053] Such aflagellate strains of H.pylori are of particular interestfor employment in serology and, as a consequence, for the in-vitrodetection of an infection due to H.pylori. These strains areadvantageously of the recombinant type.

[0054] In particular, these strains exhibit the advantage of enabling aninfection due to H.pylori to be detected in vitro in a specific andsensitive manner. In other words, the invention advantageously enablesan infection due to H.pylori to be detected specifically while avoiding,in particular, “false-positive” results, for example with bacterialstrains such as Salmonella or Campylobacter.

[0055] Given that the strains of H.pylori of the aflagellate type, whichhave thus been defined, my also have other applications, for example maybe employed in the preparation of vaccine compositions, there can beinterest in preparing recombinant aflagellate bacterial strains whichpossess a second modification or mutation, for example an aflagellatebacterial strain can be prepared which is characterized in that it isadditionally mutated in such a way that it produces an attenuatedurease, or even no longer produces urease, with the mutation consisting,for example, of a mutation of the nucleotide sequence of one or moregenes selected from among the genes ureA, ureB, ureC, ureD, ureE, ureF,ureG, ureH or ureI. The urease structural genes, designated urcA, ureB,ureC and ureD of urease, have been described in the publication (Labigneet al (1991) J. Bacteriol. 173: 1920-1931). The other genes have beendescribed in Patent Application EP 0610322.

[0056] The bacterial strains of the invention may be employed as such orin extract form, and, in particular, the invention relates to a totalbacterial strain extract which is obtained from the previously describedstrains.

[0057] Such a bacterial extract can be prepared by extracting withn-octyl glucoside. In this case, the preparation technique which isemployed is that described by LELWALA-GURUGE J. (Scand. J. Infect. Dis.1992, 24: 457-465).

[0058] Another bacterial extract can be obtained by extracting with PBSor glycine using the techniques described, respectively, by BAZILLOU M.et al (Clin. Diagn. Lab. Immuno., 1994, 1: 310-317) and AGUIRRE P. M.(Eur. J. Clin Microbiol. Infect. Dis., 1992 11: 634-639).

[0059] Within the scope of these applications, the invention relates toa composition for the in-vitro detection of an infection due to H.pyloriin a sample of biological fluid obtained from a patient, in particularin a sample of serum, which composition includes, as the activeprinciple, a bacterial strain of the invention or a bacterial extract inaccordance with the description given above.

[0060] The biological samples which are used may be of any type and can,in particular, be any type of biological fluid, such as serum, saliva orurine, for example.

[0061] In the same way, the techniques which are employed for thedetection are any techniques which involve reactions of theimmunological type, in particular of the antigen/antibody type. Forexample, use is made of techniques such as Western blot, ELISA, etc.

[0062] The invention also relates, therefore, to a method for thein-vitro detection of an infection due to H.pylori in a sample ofbiological fluid taken from a patient, in particular in a sample ofserum, which method comprises the steps of:

[0063] bringing the sample under test into contact with a bacterialstrain according to the invention or with a bacterial extract as definedabove.

[0064] detecting an immunological reaction between the said bacterialstrain and antibodies which are directed against H.pylori and which arepresent in the sample under test.

[0065] By way of example, an in-vitro detection on a biological samplein order to look for an infection due to H.pylori can be carried out byimplementing the following steps:

[0066] plates are covered with the antigen which is used for thedetection and which may be a pure or recombinant protein or else anaflagellate strain or a bacterial extract, in particular an NOG (n-octylglucoside) extract of the N6flbA- strain (by way of example, thequantity of extract might be 3 μg/ml or the quantity of antigen might be2 μg/ml),

[0067] a range of negative and positive controls (the positive controlbeing employed at differing dilutions) is used, and patient sera, whichare diluted to {fraction (1/100)}, are tested in parallel (volumedeposited, 100 μl,

[0068] an incubation step is then carried out, for example at 37° C. forone hour, which step is followed by several successive washings and by afurther incubation, for example at 37° C. for 1 hour, with a monoclonalconjugate (of the human IgS type labelled with peroxidase), whichconjugate is employed at differing dilutions (for example at a dilutionof 1/32000 in the case of an antigen and at a dilution of 1/64000 in thecase of a bacterial extract), with the deposited volume being 100 μl.

[0069] after the incubation with the monoclonal conjugate, severaldifferent washings are carried out (for Example 4) and the enzymicreaction is developed, in the dark and for 30 minutes, using“OPD+substrate”. The enzymic reaction is then stopped by adding H₂SO₄,after which the optical densities, OD's, are read at 192 nm/620 nm.

[0070] The invention is furthermore directed to an immunogeniccomposition for obtaining antibodies against H. pylori, whichcomposition is characterized in that it includes, as the activeprinciple, a bacterial strain according to the invention or an extractof this bacterial strain.

[0071] According to one particular embodiment of the invention, animmunogenic composition for obtaining antibodies against H.pylori ischaracterized in that it includes an amino acid sequence from the FlbAprotein.

[0072] Also included within the scope of the present invention is avaccinating composition for obtaining antibodies which protect againstan infection due to H.pylori, characterized in that it includes, as theactive principle, a bacterial strain according to the invention or abacterial extract according to the above definitions.

[0073] Another vaccinating composition for obtaining antibodies againstan infection due to H.pylori is characterized in that it includes, asthe active principle, antigens of the urease type, in particularantigens encoded by the genes urcA, ureB, ureC, or ureD and a proteinhaving an amino acid sequence as defined above.

[0074] The invention also relates to monoclonal antibodies or polyclonalsera which are directed against a previously described amino acidsequence. These antibodies are obtained by techniques which are knownper se, in particular by immunizing an animal with the chosen antigen,followed either by producing and recovering the antibodies which areproduced and selecting those among them which specifically recognizeH.pylori, or by preparing hybridomas, by fusing spleen cells from thepreviously immunized animal with myeloma cells, with these hybridomasthen being cultured in order to obtain monoclonal antibodies, which areselected on the basis of the specificity with which they recognize thechosen H.pylori antigen.

[0075] Other monoclonal antibodies or polyclonal sera according to theinvention are directed against an aflagellate H.pylori strain such asdescribed in the preceding pages.

[0076] The invention furthermore relates to a composition for the invitro detection of an infection due to H.pylori in a biological sample,which composition includes, as the active principle, monoclonalantibodies or a polyclonal serum which have been obtained against anH.pylori strain of the aflagellate phenotype according to the invention.

[0077] The invention also relates to nucleotide sequences, as the activeprinciple of a medicament, which encode amino acid sequences accordingto the invention, which amino acid sequences are able to induce animmunogenic response in an animal or in a patient. A technique foremploying nucleotide sequences as medicaments has been described byDONNELY et al 1995, Nature Medic. 1(6), pp. 583-587.

[0078] FIG. 1

[0079] 1A: Restriction map of the plasmid pILL570 and of the minitransposon Tn3 containing the cassette of the gene for resistance tokanamycin.

[0080] 1B: Linear restriction maps of the recombinant plasmids pSUS39and pSUS207. The numbers which are shown correspond to the sizes of therestriction fragments, expressed in base pairs, H: HINdIII; Bg: BglII.The presence of an asterisk indicates that the restriction site wasmodified during the cloning and that it is no longer recognized by thecorresponding restriction enzyme.

[0081] FIG. 2: Nucleotide sequence of the flbA of H.pylori and thededuced amino acid sequence, given in one-letter code.

[0082] FIG. 3: Multiple alignment of the FlbA protein of H.pylori withfive other members of the LcrDk/FlbF family. CjFlbA: Campylobacterjejuni FlbA; CcFlbF: Caulobacter crescentus, FlbF; YplerD: Xersiniapestis LerD; StlnvA: Salmonella typhimurium InvA; SfMxiA: Shigellaflexneri MxiA. The asterisks indicate the positions of the amino acidswhich are conserved in all the homologs of the LerD/FlbF family; thedots indicate the positions of the amino acids which are conserved in atleast 5 out of the 6 homologous proteins; the conserved amino acidsequences which were used for synthesizing the degenerateoligonucleotides (OLFlbA-1 and OLFlbA-2) are underlined. Particular noteshould be taken of the degree of conservation of the N-terminal domainof these homologous proteins, which contrasts with the degree ofvariability of the hydrophilic domain of the C-terminal region.

[0083] FIG. 4: Diagrammatic depiction of the phylogenetic tree of sixproteins belonging to the LerD/FlbF family. The proteins which areinvolved in regulating the expression of mobility, i.c. FlbA of H.pylori(HpFlbA) and of Campylobacter jejuni (CjFlbA), and FlbF of Caulobactercrescentus (CcFlbF) form a branch which is distinct from that of theproteins involved in the secretion of virulence proteins (InvA, MxiA andLerD of Salmonella, Shigella and Yersinis, respectively). The numberswhich are shown depict the relative evolutionary distance.

[0084] FIG. 5: Diagrammatic representation of the strategy which wasfollowed for constructing the isogenic mutants of H.pylori strain N6,i.e. mutants in which the gene encoding the FlbA protein was inactivatedby inserting a gene encoding for resistance to kanamycin.

[0085] FIG. 6: Analysis by immoblotting (Western blot) of the proteinsfrom an N6-flbA⁻ mutant using AK179 antiserum (3), which is specificallydirected against flagella which have been purified from H.pylori: 1:N6-flbA mutant; 2: flaA/flaB double mutant; 3: flaB (B) mutant; 4: flaA(8) mutant; 5: wild-type N6 strain.

[0086] FIGS. 7 to 11: Comparative results from the serology carried outon H.pylori.

[0087] FIGS. 12 and 13: Extractions using the aflagellate strainN6flbA-: the extractions were carried out using glycine, PRS or NOG.

[0088] FIG. 12: The curves were constructed on the basis of thefollowing data: NET ABS CALC STD# CONC 750.0 CONC DIFF COEFFS: MEAN: 10.0000 0.0020 −0.003 0.0080 P2 = 2.0324 −1.03568-07 2 0.1660 0.07600.1721 −0.006 P1 = 2.2753 3 0.3300 0.1400 0.3459 −0.016 P0 = 0 4 0.66500.2390 0.6474 0.0176 5 1.3300 0.4280 1.3336 −0.004

[0089] FIG. 13: Minimethod (BIO-RAD) protein assays Glycine: diluted ½;glucoside: diluted {fraction (1/10)}; supernatant 1: diluted ¼;supernatant 2: not diluted.

[0090] The curves were constructed on the basis of the following data:NET ABS CALC STD# CONC 750.0 CONC DIFF COEFFS: 1 0.0000 −0.003 1.5398−1.540 P2 = 144.63 2 25.000 0.0600 21.861 3.1392 P1 = 314.31 3 50.0000.1470 51.810 −1.810 P0 = 2.4815 4 100.00 0.2750 99.855 0.1454 5 200.000.5030 199.94 0.0636

EXAMPLES

[0091] I Identification of the flbA gene and preparation of aflgellatestrains

[0092] Among the proteins which are known to play a role in regulatingthe expression of bacterial mobility, the proteins belonging to therecently identified LcrD/FlbF family, which include the LerD protein ofthe bacteria of the genus Yersinia (6), the InvA protein of Salmonella(2), MxiA of Shigella (1), FlbF of Caulobacter crescentus (7) and LfbAof Campylobacter jejuni (4) are proteins of interest. The LerD, InvA andMxie proteins are involved in the regulation and/or the secretion ofproteins which are associated with the virulence of the bacteria whichexpress them, whereas the FlbF protein of Caulobacter crescentus and theFlbA protein of Campylobacter jejuni are involved in regulating thebiosynthesis of the flagella and therefore involved in regulatingmobility. The homologs of the LerD/FlbA family which are known to datepossess very conserved domains, especially in the N-terminal part ofthese proteins, and it was therefore possible to use two of theseconserved regions (MPGKQM, amino acids 151 to 156 of the LerD protein ofXersinia) and MDGAMKF (amino acids 189 to 195 of LerD) for defining twodegenerate oligonucleotides (OLFlbA-1 and OLFlbA-2, Table 1), which weresynthesized and which have served as nucleotide primers in the geneamplification experiments which were carried out on the chromosomal DNAof Helicobacter pylori. In this way, it was possible to amplify afragment of 130 base pairs (bp), and determination of its nucleotidesequence demonstrated that this fragment encoded a segment of a proteinwhich was very homologous to the proteins of the LerD/FlbF family. Thisamplified fragment was then labelled radioactively and used as a probeto screen an H.pylori cosmid library.

[0093] This fragment corresponds to the sequence contained betweennucleotides 575 and 707 of the sequence depicted in FIG. 2.

[0094] One of the cosmids of the genomic library was identified asencoding the LerD/FlbF homolog of H.pylori and was then subjected to apartial digestion with Sau3A so as to construct a mini library (200subclones) of the cosmid in vector pILL570, containing insertedfragments possessing a size of between 2 and 5 (kilobases). VectorpILL570 has been described in the paper by Labigne A. et al (InstitutPasteur/Elsevier Paris 1992. Rec. Microbiol. 1992, 143, 15-26). Itsrestriction map is given in FIG. 1A. These 200 clones were thenhybridized to the 130 bp probe, and the clones which harboured plasmidspSUS39 and pSUS207 gave a positive hybridization. The linear restrictionmaps of these two recombinant plasmids are depicted in FIG. 13 anddemonstrate that the two inserts of these clones have overlappingsequences. Determination of the nucleotide sequences of these twoinserts revealed that neither of the two inserts contained the flbA genein its entirety. The flbA gene of H.pylori, designated in this way dueto its homology with the flbA gene of Campylobacter jejuni, correspondsto an open reading frame of 2196 nucleotides and encodes a proteinhaving a calculated molecular mass of 80.1 kilodaltons. The mucleotidesequence of flbA and the amino acid sequence of FlbA are given in FIG.2. Consensus sequences which are characteristic for promoter orterminator sequences have not been detected upstream and downstream ofthe open reading frame.

[0095] The FlbA protein exhibits similarities with the FlbA protein ofCampylobacter jejuni and the FlbF protein of Caulobacter crescentus,both of which are involved in mobility (51.7% and 40.4% identity,respectively) whereas these percentages are lower with members of theLcrD/FlbF protein family which are not involved in mobility: 32.8%identity with LerD from Yersinia, 30.5% with MxfA from Shigella and29.3% with InvA from Salmonella. A multiple alignment of the amino acidsequences of these proteins with that of H.pylori FlbA is given in FIG.3. The most conserved regions of the homologs of the LerD/FlbF familyare located in the N-terminal part of the proteins.

[0096] The phylogenetic evolution of the proteins involved in mobility(FlbA and FlbF) and that of the proteins involved in regulating theexpression and/or the secretion of proteins associated with virulence isdepicted diagrammatically by a phylogenetic tree (FIG. 4). Two distinctbranches can be seen: H.pylori FlbA belongs unambiguously to the branchcorresponding to the regulatory proteins involved in the biosynthesis ofthe flagella.

[0097] Construction and characterization of isogenic mutants of H.pylori which are deficient in the synthesis of the FlbA protein.

[0098] A 1600 base pair fragment was amplified from plasmid pSUS39 usingthe oligonucleotides OLFlbA-7 and OLFlbA-8 (Table 1), each of whichcontains a BamHI restriction site at its 5′ end. In its central region,this amplified fragment contains a unique HindIII restrictionendonuclease site and was cloned into vector pSUS33, which is aderivative of plasmid pUC19 in which the HindIII site situated in themultiple cloning site has been deleted. In order to obtain pSUS33,plasmid pUC19 was restricted with HindIII; the sticky ends resultingfrom this restriction were treated with Klenow enzyme and T4 DNApolymerase in order to produce blunt ends; the resulting fragment wasreligated with T4 DNA ligase and introduced into E. coli DH5x in orderto produce pSUS33. The recombinant plasmid resulting from theintegration of the 1600 base pair fragment into pSUS33 was designatedpSUS40; it was linearized with HindIII, its ends were blunt-ended andthe SmaI kanamycin cassette, which was derived from plasmid pILL600(Labigna A. et al, 1988, J. Bacter. 170, 1704-1708), was cloned intothis unique site, resulting in plasmid pSUS42. Plasmid pSUS42 was thenintroduced by electroporation into the “N6” strain of H.pylori. Theelectroporation was carried out in accordance with the techniquedescribed by Perrero R. L. et al (Journal of Bacteriology, July 1992,pp. 4212-4217, Vol. 174, No. 13). The transformants which were obtainedafter selecting on a selective medium containing kanamycin (25 μg/ml)were then characterized genotypically and phenotypically. FIG. 5 shows adiagram of the procedure which was followed for the construction ofmutants. Genotypic characterization of these mutants, by geneamplification and Southern hybridization, demonstrated that the genomesof the transformants which were resistant to kanamycin contained theresistance gene inserted in the middle of the flbA gene and that therehad therefore been an allelic replacement, by means of doublecrossing-over, of the wild-type copy of the flbA gene by the inactiveflbA-Km copy, with the loss of the nucleotide sequences of the pSUS33vector. Phenotypic characterization of the flbA ⁻ mutants of H.pyloridemonstrated that they were not mobile; furthermore, analysis of thesemutants by electron microscopy revealed that there was a total absenceof the flagellum elements and an absence of the flagellum sheath. Theimmunoblotting experiments (Western blots) which were carried out usingantibodies directed against the proteins of the entire flagellum ofH.pylori (FIG. 6) demonstrated that two peptide bands corresponding tothe flagellar subunits FlaA and FlaB were absent, as was a bandcorresponding to a polypeptide of an apparent mass of 90 kilodaltons,which is a protein which has recently been identified by O'Toole andcollaborators (5) as being the hook protein (or anchoring protein) ofthe flagellum (5).

[0099] Taken as a whole, these results suggest that the FlbA protein ofH.pylori is essential for the biosynthesis of all the flagellarstructures and that inactivation of the gene encoding this proteinresults in complete cessation of the synthesis of any structure enteringinto the formation of the flagellum and not in interruption of theexport of the constituents of these structures. TABLE 1 Oligonucleotidesemployed in this study Oligo- nucleotide Position Attand Nucleotidesequence OLF1bA-1 AG-151-155 (LerD) + ATGCCTCGAGGTCGAAAAGCAACATGOLF1bA-2 AS-189-195 (LerD) − GAAATCTTCATACTGGCAGCTCCAGTC OLF1bA-7515-534 + CGGGATCCGTCGTTACTAATGGTTCTAC OLF1bA-8 2092-2111 −CGGGATCCTCATGGCCTCTTCACAGACC

[0100] II H.pylori serology Models studied 1) HspAmr1E recombinantprotein of 47.5 kD (HspA = 13 kD) A sensitivity of 41% and a speci-ficity of 96% were obtained on the population termed population 1 ofdocumented sera. 2) N6flbA- aflagellate strain of Helicobacter pylori 3extractions were carried out: - n-Octyl glucoside - PBS - Glycine Forthe time being, the extrac- tion with n-octyl glucoside. (NOG) appearsto be the best. 3) -N6 corresponding wild-type strain An extraction wascarried out with n-octyl glucoside.

[0101] A second population of sera was employed (population II). Thispopulation consists of some one hundred sera which are well documentedfrom the clinical, endoscopic, histological, bacteriological andanatomopathological points of view. It was this population II which wasused to assess the performances of the different models under study.Five different populations were tested.

[0102] 5 populations of tested scra:

[0103] 300 ordinary sera (FNTS)

[0104] 18 sera which were positive by WHITTAKER serology (CBMS)

[0105] 92 well documented sera termed sera of population II

[0106] 87 sera which were documented from the bacteriological andanatomopathological points of view and which were termed sera ofpopulation I.

[0107] 23 sera exhibiting cross reactions:

[0108] 10 anti-Legionella positive sera

[0109] 10 anti-Chlamydia positive sera

[0110] 3 anti-Campylobacter positive sera

[0111] Two competing kits, which bibliographic studies indicated wereeffective, were tested in parallel.

[0112] 2 tested commercial kits:

[0113] Cobas Core (ROCHE)

[0114] Pylori Stat (WHITTAKER)

[0115] Results

[0116] The ordinary sera (FNTS) (FIGS. 8 to 11, Table 2)

[0117] 300 sera were taken through the following models:

[0118] Hsp A malE

[0119] N6 flbA-

[0120] N6

[0121] The epidemiological studies give seroprevalences, in France, ofbetween 20 and 25%. The distribution of 300 blood donor sera was studiedand the prevalence of positivity was calculated for different thresholdvalues in order to validate the threshold value which was previouslydefined using the CBMS serum library (WHITTAKER serology).

[0122] this study enables the different tests to be compared using thesame seroprevalence.

[0123] The first 43 sera were also taken through the following models:

[0124] Cobas Core (ROCHE)

[0125] Pylori Stat (WHITTAKER)

[0126] serology known as JLF serology (ELISA test, based on an aqueousextract of several bacterial strains)

[0127] The results are expressed in arbitrary units and for differentthreshold values; a positive result is written as 1 and a negativeresult is written as 0.

[0128] On comparing these 43 sera in different tests, it can be observedthat:

[0129] the aflagellate strain N6flbA- and the Cobas Core test (Roche)give comparable seroprevalences of the order of 20%.

[0130] HspA gives a very low seroprevalence (7%), which suggests a lackof sensitivity in view of the subsequent results.

[0131] the JLF serology appears to be very specific since theseroprevalence is only 14%, considering the subsequent results.

[0132] the Pylori Stat test (Whittaker) gives a high seroprevalence(29%), which might indicate a lack of specificity or a threshold valuewhich is too low. TABLE 2A Comparison of 43 FNTS nora with regard to:No. HspA 150 C30 + h C10 + h C. Core P P. Stat 0.38 PBS 100 80 60 NOG100 30 80 0.30 NOG 100 1 0 0 1 1 170 1 0.70 3393 1 1 1 3105 1 1 1 0.751 >928 1 2 0 0 0 0 0 0 0.21 0 4 0 0 0 3 0 0 0 0.01 0 5 0 3 0 0 0 0 3 00.23 0 0 0 0 0 4 0 0 0 0.01 0 0 0 4 0 0 0 0 4 0 0.19 0 4 0 0 0 3 0 0 00.02 0 4 0 5 0 0 0 0 3 0 0.20 0 12 0 0 0 0 0 0 0 0.08 0 27 0 6 1 0 0 0 20 0.17 0 0 0 0 0 0 0 0 0 0.01 0 0 0 7 2 0 0 0 3 0 0.32 0 41 0 0 0 39 0 00 0.02 0 13 0 8 79 0 0 0 137 1 3.37 1 0391 1 1 1 3109 1 1 1 1.43 1 >9281 9 0 0 0 0 3 0 0.35 1 23 0 0 0 21 0 0 0 0.06 0 18 0 10 0 0 0 0 1 0 0.340 28 0 0 0 14 0 0 0 0.02 0 31 0 11 0 0 0 0 3 0 0.23 0 81 0 0 1 33 0 0 00.00 0 77 0 12 0 0 0 0 0 0 0.19 0 0 0 0 0 0 0 0 0 0.02 0 0 0 13 0 0 0 01 0 0.19 0 31 0 0 0 28 0 0 0 0.02 0 37 0 14 0 0 0 0 3 0 0.25 0 4 0 0 0 40 0 0 0.02 0 11 0 15 1 0 0 0 3 0 0.27 0 4 0 0 0 3 0 0 0 0.02 0 7 0 16 00 0 0 0 0 0.37 1 02 0 0 1 31 0 0 0 0.05 0 97 0 17 0 0 0 0 1 0 0.22 0 3 00 0 2 0 0 0 0.03 0 13 0 18 0 0 0 0 1 0 0.25 0 13 0 0 0 0 0 0 0 0.00 0 180 19 0 0 0 0 0 0 0.27 0 4 0 0 0 4 0 0 0 0.08 0 15 0 20 0 0 0 1 0 0.21 023 0 0 0 12 0 0 0 0.01 0 31 0 21 0 0 0 0 18 1 0.40 1 34 0 0 0 204 1 1 10.24 0 331 1

[0133] TABLE 2B Comparison of 43 FNTS nern with regard to: No. HspA 150C30 + h C10 + h C. Core P P. Stat 0.38 PBS 100 80 60 NOG 100 30 80 0.30NOG 100 22 — 0 0 0 1 0 3.43 1 13 0 0 0 40 0 0 0 0.05 0 21 0 23 0 0 0 125 1 0.49 1 285 1 1 1 298 1 1 1 0.37 1 >928 1 24 9 0 1 1 125 1 0.65 13390 1 1 1 3100 1 1 1 1.47 1 >928 1 25 0 0 3 0 0 0 0.20 0 34 0 0 0 20 00 0 0.01 0 51 0 26 0 0 0 0 2 0 0.28 0 97 0 1 1 60 8 0 0 0.04 0 105 1 270 0 1 1 10 1 0.33 0 285 1 1 1 239 1 1 1 0.07 0 359 1 28 0 0 1 1 7 0 0.210 28 0 0 0 14 0 0 0 0.05 0 27 0 29 3 0 0 0 8 0 0.20 0 3 0 0 0 2 0 0 00.01 0 0 0 30 1 0 0 0 2 0 0.23 0 3 0 0 0 3 0 0 0 0.01 0 4 0 31 0 0 0 0 10 0.21 0 0 0 0 0 1 0 0 0 0.03 0 0 0 32 0 0 0 0 2 0 0.31 0 24 0 0 0 15 00 0 0.75 0 30 0 33 0 0 0 0 1 0 0.23 0 14 0 0 0 8 0 0 0 0.02 0 8 0 34 0 00 0 3 0 0.23 0 0 0 0 0 1 0 0 0 0.00 0 2 0 35 1203 1 1 1 170 1 0.874 1403 1 1 1 3103 1 1 1 0.08 1 >928 1 36 0 0 0 0 4 0 0.38 1 41 0 0 0 32 0 00 0.04 0 00 0 37 0 0 0 0 5 0 0.22 0 110 1 1 1 100 1 1 1 0.02 0 227 1 3813 0 0 0 4 0 0.47 1 11 0 0 1 60 0 0 1 0.07 0 108 1 38 1 0 0 0 4 0 0.34 023 0 0 0 13 0 0 0 0.05 0 34 0 39 1 0 0 0 4 0 0.34 0 23 0 0 0 13 0 0 00.05 0 34 0 40 0 0 0 0 2 0 0.19 0 3 0 0 0 8 0 0 0 0.02 0 4 0 41 0 0 0 00 0 0.24 0 48 0 0 0 23 0 0 0 0.05 0 88 0 42 0 0 0 0 170 1 0.59 1 3380 11 1 3104 1 1 1 1.47 1 >928 1 43 0 0 1 1 3 0 0.19 0 7 0 0 0 3 0 0 0 0.040 8 0 no. of 8 7 8 12 8 9 12 0 0 10 0 11 % of 1% 14% 18% 19% 29% 19% 21%28% 21% 21% 23% 14% 28%

[0134] The sera which are positive by WHITTAKER serology (CBMS) (Table3)

[0135] Three sera were found to be positive only with the Pylori Stattest (Whittaker). They were not confirmed using any other test.

[0136] It may be supposed that this result is due to this test lackingspecificity. If the Cobas Core test (Roche), which is one of the bestwhich is currently on the market, is taken as the reference, we cancompare our different models in relation to Cobas Core.

[0137] The aflagellate N6flbA- strain correlates perfectly with CobasCore.

[0138] The 3 sera which are negative with Cobas Core are also negativewith N6flbA-.

[0139] The 15 sera which are positive with Cobas Core are also positivewith N6flbA-.

[0140] The wild-type N6 strain gives the same results as the aflagellatestrain.

[0141] HspA also lacks sensitivity since 9 Cobas Core-positive sera arenegative with HspA.

[0142] The 3 sera which are negative with Cobas Core are also negativewith HspA.

[0143] The sera of population II

[0144] 92 sera were selected with the sera dividing into 3 groups:

[0145] 34: dyspeptic patients diagnosis of ulcer (duodenal or gastric)by endoscopy and histology presence of Helicobacter pylori, by cultureand/or anatamopathologically; a rapid urea test was also carried out.This group will be termed Hp+/U+

[0146] 27: dyspeptic patients differential diagnosis of ulcer (gastritisetc.) by endoscopy and histology presence of Helicobacter pylori byculture and/or anatamopathologically; a rapid urea test was also carriedout. This group will be termed Hp+/U−

[0147] 31: patients which are or are not dyspeptic normal gastroduodenumby endoscopy and histology absence of Helicobacter pylori by culture andanamatopathologically; a rapid urea test was also carried out. Thisgroup will be termed Hp−

[0148] The clinical, endoscopic, histological, bacteriological andanatomopathological findings are indicated for each patient. This welldocumented population enabled criteria of sensitivity and specificity tobe defined.

[0149] HpA: A substantial lack of sensitivity, as observed withpopulation I, is still noticed. The sensitivity is 59%, with aspecificity of 100.

[0150] N6flbA: A sensitivity of 100% is confirmed for the n-octylglucoside extract, with a specificity of 90%. This result is comparableto that obtained with the Roche Cobas Core test (98% sensitivity with aspecificity of 94%).

[0151] N6: On population II, the wild-type strain is entirely comparableto the aflagellate strain. None of the 31 negative sera is positive withthe wild-type strain; no cross reaction due to the flagellum wasdetected with this population II.

TABLE 6 Sera of population II In relation to the presence of Hp (cultureand/or anamatopathologically) and ulcer Sensibility Specificity Inrelation N6flbA HspA malE VS = 100 44.1% (15/34) 100% (31/31) to Hp+ andVS = 50 52.9% (18/34) 100% (31/31) DU/GU VS = 20 64.7% (22/34) 73.8%(25/31) that is: NOG VS = 100 94.1% (32/34) 96.8% (30/31) 34Hp+/U+ VS =80 94.1% (32/34) 93.6% (29/31) VS = 60 100% (34/34) 90.3% (28/31) PBS VS= 100 62.4% (28/34) 93.6% (29/31) VS = 80 94.1% (32/34) 93.6% (29/31) VS= 60 97.1% (33/34) 83.9% (26/31) JLF Sero VS = 0.30 62.4% (28/34) 96.8%(30/31) Pylori Stat 94.1% (32/34) 90.3% (28/31) Cobas Core 100% (34/34)93.6% (29/31)

[0152] TABLE 7 Sera of population II In relation to the presence of Hp(culture and/or anamatopathologically) Specificity Sensibility In HspAVS = 100 45.9% (28/61) 100% (31/31) relation malE VS = 50 59% (36/61)100% (31/31) to VS = 20 80.7% (45/61) 73.8% (25/31) Hp+: N6flbA- NOG VS= 100 95.1% (58/61) 96.8% (30/31) −34 VS = 80 95.1% (58/61) 93.6%(29/31) DU/GU VS = 60 100% (61/61) 90.3% (28/31) −27 PBS VS = 100 85.3%(52/61) 93.6% (29/31) GNU VS = 80 93.4% (57/61) 93.6% (29/31) that is VS= 60 96.7% (59/61) 83.9% (26/31) 61 Hp+ JLF sero VS = 0.30 78.7% (48/61)96.8% (30/31) 31 Hp− Pylori Stat 93.4% (57/61) 90.3% (28/31) Cobas 93.3%(60/61)* 93.6% (29/31) Core

[0153] TABLE 8 Sera of population II In relation to the presence of Hp(culture and/or anamatopathologically) and the absence of an ulcerSpecificity Sensibility In HspA VS = 100 48.2% (13/27) 100% (31/31)relation malE VS = 50 66.7% (18/27) 100% (31/31) to Hp+ VS = 20 85.2%(23/27) 73.8% (25/31) and Nbf1bA- NOG VS = 100 96.3% (26/27) 96.8%(30/31) GNU VS = 80 93.6% (26/27) 93.6% (29/31) that is VS = 60 100%(27/27) 90.3% (28/31) 27 Hp+/ PBS VS = 100 88.9% (24/27) 93.6% (29/31)U− VS = 80 92.6% (25/27) 93.6% (29/31) VS = 60 96.3% (26/27) 83.9%(26/31) JLF sero VS = 0.30 74.1% (20/27) 96.8% (30/31) Pylori stat 92.6%(25/27) 90.3% (28/31) Cobas 96.3% (26/27) 93.6% (29/31) Core

[0154] The place of serology

[0155] Serology is placed at 2 levels:

[0156] Very sensitive serology: for the purpose of detecting thepresence of the bacterium in young subjects complaining of epigastricpains. If the serology turns out to be negative, the subject will nothave to suffer endoscopy or a biopsy and another cause for his painswill be sought.

[0157] Risk-specific serology: this involves demonstrating the risk ofhaving a serious infection with Helicobacter pylori, that is an ulcer, acancer or a gastric lymphoma (MALT lymphoma).

[0158] either using a molecule which is specific for the risk inquestion

[0159] or using a risk-specific threshold (threshold value which ishigher in subjects which are at risk than in subjects which are not atrisk).

[0160] This specific serology can be employed to screen the generalpopulation and thus to detect cancers and lymphomas which are associatedwith Helicobacter pylori and which would not be detected because of alack of symptoms. (Only subjects which complain of pain will consult agastroenterologist).

[0161] The response to the sensitivity issue is good. TABLE 9 Mean andstandard deviation of the A.U.'s in the 3 groups of patients Hp+/U+ Hp−(n = 31) Hp+/U− (n = 27) (n = 34) Hsp A mean 10.61 775.72 770.32standard 8.81 1312.56 1666.52 deviation N6flBA-(NOG) mean 17.16 895.50944.85 standard 26.69 818.57 915.27 deviation

[0162] TABLE 10 Mean and standard deviation of the A.U.'s in terms ofgastric histology Atrophy Inflmmation Activity Intensity Hsp A NOG P.Stat Cag A Hsp A NOG P. Stat Hsp A NOG P. Stat 0 Mean 577 712 0.31standard deviation 2052 680 0.08 1 Mean 410 412 0.28 93 437 877 0.30 479938 0.32 standard deviation 1004 300 0.07 122 889 400 0.00 1117 878 0.09Mean 423 730 0.30 188 .31 733 798 0.31 standard deviation 984 707 0.08200 1055 780 0.09 1382 753 0.07 Mean 1321 1403 0.38 684 0.43 1302 14020.38 standard deviation 2059 1012 0.08 807 1742 1132 0.08 1029 1174 0.12Distribution Atrophy Inflammation Activity 0 0 0 15 1 10 21 25 2 28 3312 3 22 1 9 4 1 0 0

[0163] Correlation between the intensity of the gastritis and theantibody levels

[0164] The gastritis is defined by 3 parameters:

[0165] Atrophy (represented by the first figure after G); its intensityis marked from 1 to 4.

[0166] The global inflammation corresponds to infiltration withneutrophilic polynuclear cells and with monocytes; (represented by thesecond figure after the G). Its intensity is marked from 1 to 3.

[0167] Activity corresponds to the number of neutrophilic polynuclearcells (represented by the third figure after the G); its intensity ismarked from 0 to 3. Some folicular forms are marked F.

[0168] Normally, the following correlation can be observed:

[0169] The activity correlates very well with Helicobacter pylori.

[0170] The inflammation correlates well with Helicobacter pylori.

[0171] The means of the titren observed in each group have thereforebeen calculated in terms of these 3 parameters and their intensity.

[0172] Interpretation of the results:

[0173] Use of a t test makes it possible to demonstrate whether adifference between 2 observed means is significant or not with a 5%risk.

[0174] The hypothesis on which the t test is based is the equality ofvariances, demonstrated by an F test (Fisher test).

[0175] Since some variances are not equal, it is not therefore possibleto compare all the means with each other.

[0176] By comparing the means, when possible, it has been possible todemonstrate whether the differences between the different groups aresignificant or not.

[0177] Significant difference:

[0178] Between the means of “2” and “3” for HspA and NOG in the“Inflammation” group.

[0179] Non-significant difference:

[0180] With regard to activity, no significant differences weredemonstrated between the different intensity levels:

[0181] HspA:

[0182] no significant difference between levels

[0183] 0 and 2

[0184] 0 and 3

[0185] 1 and 2

[0186] 1 and 3

[0187] 2 and 3

[0188] NOG:

[0189] no significant difference between levels

[0190] 0 and 1

[0191] 0 and 2

[0192] 1 and 2

[0193] 1 and 3

[0194] 2 and 3

[0195] It is nevertheless possible to observe a tendency for the titresto increase in dependence on the intensity of the gastritis:

[0196] with regard to atrophy, the means double, for HspA and for theNOG extract of the aflagellate strain, when passing from level 1 to 2and from level 2 to 3.

[0197] with regard to inflammation, the means double when passing fromlevel 1 to 2.

[0198] The numbers in each group are relativity low (in each case <30)for drawing conclusions with regard to statistically significantdifferences. TABLE 11 Means of the A.U.'s in terms of gastric histology.Aatrophy Inflammation Activity Intensity Hsp A NOG P. Stat Hsp A NOG P.Stat Hsp A NOG P. Stat 0 Mean 1392 875 0.32 (standard 2610 097 0.09deviation) 1 Mean 121 328 0.75 438 311 0.30 839 1818 0.34 (standard 118218 0.05 750 431 0.08 1298 1050 0.10 deviation) 2 Mean 304 793 0.32 883848 0.33 319 827 0.32 (standard 507 784 0.09 1820 813 0.09 329 835 0.09deviation) 3 Mean 2904 1722 0.41 2133 2184 0.43 811 1318 0.38 (standard2858 1080 0.07 1989 1008 0.09 1502 1040 0.12 deviation) DistributionAtrophy Inflammation Activity 0 0 0 8 1 7 10 13 2 17 19 8 3 9 5 8 4 1 00

[0199] Sera able to exhibit cross reactions

[0200] 2 types of sera were employed.

[0201] 20 sera (10 anti-Legionella+and 10 anti-Chlamydia +) being ableto exhibit cross reactions with HspA, because these 3 bacterial possessheat shock proteins which are very akin to each other.

[0202] 3 anti-Campylobacter positive sera, in order to demonstrate crossreactions with the flagellate strain N6 which would disappear with theaflagellate strain N6flbA-. It is very difficult to obtainanti-Campylobacter positive sera; this is the reason for there onlybeing 3 sera.

[0203] HspA does not exhibit any cross reaction, either with the 10anti-Legionella positive sera or with the 10 anti-Chlamydia positivesera.

[0204] While some of these sera have positive titres ofanti-Helicobacter pylori antibodies, both with the flagellate strain andwith the aflagellate strain, the clinical contest of these sera is notknown. TABLE 12 Sera which are able to exhibit cross reactions Titer NSVS = 100 NSNBA− VS = 60 HspA VS = 100 Legionella+ A P2P3 = 256 0 0 4 047 0 B P4 P5 = 64 >528 1 641 1 42 0 C P2 P3 = 128 212 1 87 1 68 0 D P2P3 = 64 70 0 19 0 15 0 E P1 = 256/P2 = 512 >523 1 239 1 258 1 F P2 P3 P4P5 = 128 322 1 121 1 41 0 G P1 = 512/P5 = 1024 >923 1 193 1 121 1 H P4P5 = 64 >928 1 479 1 18 0 I P2 = 128/P3 = 64 33 0 17 0 25 0 Chlamydis+ A256 5 0 8 0 25 0 B 56 7 0 9 0 34 0 C 64 535 1 290 1 39 0 D 255 357 1 2251 19 0 E 32 >928 1 855 1 19 0 F 128 >928 1 783 1 27 0 G 32 115 1 55 0 150 H Twar 16 19 0 10 0 14 0 I 32 >528 1 592 1 >928 1 J Twar 64 610 1 2801 44 0 Campylobacter+ A 35 0 28 0 17 0 B 13 0 4 0 27 0 C 50 0 68 1 89 0

CONCLUSION

[0205] HspA malE

[0206] It is still not possible to use this molecule on its own since italso lacks sensitivity, but it could be of interest if it is associatedwith other molecules.

[0207] It nevertheless carries a risk of cross reactions due to thesubstantial conservation of these heat shock proteins between thedifferent bacterial species.

[0208] N6flbA-

[0209] This aflagellate variant appears to be of great interest; thesensitivity and specificity which were obtained with serum population IIdemonstrate a very favourable efficacy.

[0210] N6

[0211] For the time being, the flagellate strain appears to be ofinterest. However, the cross reactions relating to the flagellum haveonly been studied to a limited extent due to the difficulty of obtainingsera which are well documented with regard to Campylobacter serology.

[0212] JLF test

[0213] A serological test based on an aqueous (PBS) extract of severalstrains of Helicobacter pylori was developed. This test appears to bevery efficacious.

[0214] A NOG extract of the aflagellate variant was used to test serumpopulation I.

[0215] 87 sera, which were documented only from the bacteriological andanatomopathological points of view, were tested with the aflagellatebacterial extract.

[0216] A serum is positive if the culture is positive or if theanatomopathology and the rapid urea test are positive.

[0217] A serum is negative if the 3 tests (culture, anatomopathology andrapid urea test) are negative.

[0218] A sensitivity of 90.3% (28/31) is found together with aspecificity of 71.4% (40/56).

[0219] Of 16 sera which are falsely positive using a first test, 9 arepositive either using JLF serology or using the JLF Western blot, orusing both of them.

[0220] Of the 3 sera which are falsely negative using a first test, all3 are negative either with JLF serology or with JLF Western blot, andone serum is negative with both the systems.

TECHNIQUE

[0221] Plates coated with:

[0222] HspA antigen at 2 μg/ml

[0223] NOG extract of NflbA and N6 at 3 μg/ml

[0224] Range:

[0225] 5 range points negative control

[0226] positive control used at 4 dilutions

[0227] Patient sera:

[0228] {fraction (1/100)} dilution

[0229] volume deposited: 100 μl

[0230] Incubation: 37° C. for 1 hour

[0231] 3 washings:

[0232] Monoclonal conjugate (IgG toxo)

[0233] used at

[0234] 1/32,000 for HspA

[0235] 1/64,000 for N6flbA-

[0236] 1/56,000 for N6

[0237] volume deposited:

[0238] 100 μl

[0239] Incubation of the conjugate: 37° C. for 1 hour

[0240] 4 washings

[0241] Development of the enzyme reaction using OPD+substrate

[0242] 30 minutes in the dark

[0243] Termination of the enzyme reaction with H₂SO₄

[0244] Reading of the OD at 492 nm/620 nm

[0245] Conversion of the OD's into arbitrary units (AU).

TABLE 17 Documented population from population I 55 Hp− sera 42 Hp+ seraSENSITIVITY SPECIFICITY JLF sero 85.7% (36/42) 70.9% (39/55) NOG 6097.6% (41/42) 61.8% (34/55)

[0246] EXTRACTION PROTOCOLS USING THE AFLAGELLATE STRAIN N6flba-.

[0247] Quantity supplied: 800 mg of bacteria collected using PBS andcentrifuged.

[0248] 3 extractions tested.

EXTRACTIONS OF THE AFLAGELLATE STRAIN

[0249] Clycin- n-octyl glucoside PBS extraction extraction extractionRecovery PBS 0.01M PBS PHS, pH 7.4 Washing Twice in PPS; Twice in PBS;8000 rpm/12 min 8000 rpm/12 min Extraction 0.2M acid glycine PBScontaining: Vorenx for buffer, pH 2.2. 1% n-octyl glucocide, 1 min. for15 min and at pH 7.2 room temperature (Sigma Chemical Co.), gentleagitation for 20 min at room 100 mg temperature (wgt weight) per 2.5 mlCentrifugation 11,000 g for 23,500 g for 5,000 g for 15 min 20 min 10min Neutralization 1M NaOH Dialysis PBS, pH 7.2, PBS, pH 7.7, for PBS,pH 7.2, for 24 h at +4° C. 24 hours at +4° C. for 24 h cut-off: 10,000cut-off: 10,000 at +4° C. cut-off: 10,000 Storage determination ofremoval of the determina- the concentration insoluble particles tion ofthe storage at −20° C. storage at −20° C. concentra- tion storage at−20° C.

SDS PAGE ON DIFFERENT EXTRACTS OF THE AFLAGELLATE STRAIN N6 FLBA-

[0250] SDS PAGE ON DIFFERENT EXTRACTS OF THE AFLAGELLATE STRAIN N6 FLBA-Well Concentration Sample volume/ Volume No. Sample Type μg/ml buffervolume loaded 1 MW standard 5 + 5/190 10 2 Glycine extract 202.9 60/6060 3 4 n-octyl 874 51/39 60 glucoside extract 5 6 PDS 1 extract 539.260/20 60 7 8 PBS 2 extract 77.9 60/20 60 9 10 MW standard 5 + 6/190 1011 Glycine extract 2770.7 20/20 20 pellet 12 13 Glucoside 972.9 40/40 60extract pellet 14 15 Sedimented 309.3 60/20 60 glycine extract 16 17HspA Mal E 3000 20/20 20 18 19 20 Zaleidoscope 20

[0251] References:

[0252] 1. Andrews, G. P., Maurelli, A. T.: mxiA of Shigella flexneri 2a,which facilitates export of invasion plasmid antigens, encodes a homologof the low-calcium-response protein LcrD of Yersinia Pestis. Infect.Immun. 60: 3287-3295 (1992).

[0253] 2. Galan, J. E., Ginocchio, C. Cosleas, P.: Molecular andfunctional characterization of the Salmonella invasion gene invA:homology of InvA to members of a new protein family. J. Bacteriol. 174,4338-4349 (1992.

[0254] 3. Leying, H., Suerbaum, S. Geis, G., Haas, R.: Cloning andgenetic characterization of a Helicobacter pylori flagellin gene. Mol.Microbiol. 6. 2563-2874 (1993).

[0255] 5. O'Toole, P. W., Kostrzynska, M., Trust. T. J. : Non-mobilemutants of Helicobacter pylori and Helicobacter muslelae defective inflagellar hook production. Mol. Microbiol. 14, 691-703 (1994).

[0256] 6. Plano, G. V., Barve, S. S., Straley, S. C.: LcrD, amembrane-bound regulator of the Yersinia pestis low-calcium response. J.Bacteriol. 173. 7293-7303 (1991).

[0257] 7. Ramakrishnaan, G., Zhao, J- L., Newton A.: The cellcycle-regulated gene fibF of Caulobacter crescentus is homologous to avirulence locus of Yersinia pestis. J. Bacteriol. 173, 7283-7292 (1991).

[0258] 8. Suerbaum. S., Josenhans. C., Labigne. A.: Cloning and geneticcharacterization of the Helicobacter pylori and Helicobacter mustelaeflab flagellin genes and construction of H. pylori flaA- andflaB-negative mutants by electroporation-mediated allelic exchange. J.Bacteriol. 175, 3278-3288 (1993).

1. Nucleotide sequence which regulates the biosynthesis of the flagellarproteins of Helicobacter pylori and is able to hybridize, underconditions of high stringency, with a probe corresponding to anucleotide fragment from H.pylori which has been amplified using twooligonucleotides having the following sequences: OLFlbA-1:ATGCCTCGAGGTCGAAAAGCAAGATG OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTC, orable to hybridize, under conditions of high stringency, with theseoligonucleotides.
 2. Nucleotide sequence of the flbA gene whichregulates the biosynthesis of the flagellar proteins of Helicobactorpylori, such as obtained by the steps of: screening a genomic librarycontaining the chromosomal DNA of an H.pylori strain with a probecorresponding to a nucleotide fragment from H.pylori which has beenamplified using two nucleotides having the following sequence: OLFlbA-1:ATGCCTCGAGGTCGAAAAGCAAGATG OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTC, orable to hybridize, under conditions of high stringency, with theseoligonucleotides, recovering the DNA sequences which hybridize with thesaid probe, subcloning the DNA sequences which have been obtained in anappropriate vector of the plasmid type and selecting those modifiedvectors which hybridize, under conditions of high stringency, with theprobe corresponding to the DNA fragment from H.pylori which has beenamplified using oligonucleotides OLFlbA-1 and OLFlbA-2, sequencing theDNA fragments contained in the plasmid vectors which hybridize with theabovementioned probe and determining the open reading frame contained inthese fragments.
 3. Nucleotide sequence according to claim 1 or claim 2,characterized in that it has the nucleotide sequence depicted in FIG. 2.4. Nucleotide sequence according to any one of claims 1 to 3,characterized in that it encodes a protein having the amino acidsequence depicted in FIG. 2 or an amino acid sequence possessing thesame regulatory properties with regard to the biosynthesis of theflagellar proteins of H.pylori as does the abovementioned sequence. 5.Nucleotide sequence according to any one of claims 1 to 5, whichsequence is modified, by deletion, substitution or insertion of bases orof a fragment of a nucleotide sequence, such that the flbA gene is nolonger expressed in a host cell or such that expression of the flbA genein a host cell does not enable the flagella of H.pylori to bebiosynthesized and, where appropriate, does not enable the hook proteinof H.pylori to be synthesized.
 6. Nucleotide sequence corresponding to afragment of the flbA according to any one of claims 1 to 4,characterized in that it in a fragment of at least 6 nucleotides,preferably of at least 100 nucleotides, which is derived from the flbAgene, preferably delimited by restriction sites which are present in thesequence of the flbA gene.
 7. Recombinant nucleic acid, characterized inthat it comprises a nucleotide sequence according to any one of claims 1to 6, which sequence is modified by the insertion of a cassettecontaining a marker, for example a gene for resistance to an antibiotic,or a gene for resistance to a heavy metal.
 8. Recombinant nucleic acidaccording to claim 7, characterized in that the nucleotide sequenceaccording to any one of claims 1 to 6 is modified by the insertion of acassette for resistance to kanamycin.
 9. Oligonucleotides, characterizedin that they are specific for a sequence according to any one of claims1 to 3 and in that have one of the following sequences: OLFlbA-1:ATCGTCGAGGTCGAAAAGCAAGATG OLFlbA-2: GAAATCTTCATACTGGCAGCTCCAGTCOLFlbA-7: CGGGATCCGGGTTACTAATGGTTCTAC OLFlbA-8:CGGGATCCTCATGGCCTCTTCAGAGACC
 10. Amino acid sequence of the flbA proteinof H.pylori, characterized in that it is encoded by a nucleotidesequence according to either of claims 1 and
 2. 11. Amino acid sequence,characterized in that it is the FlbA protein of H.pylori, having thesequence depicted in FIG. 2, or in that it is a fragment of this proteinwhich is recognized by antibodies directed against the FlbA protein. 12.Helicobacter pylori bacterial strain, characterized in that it has anaflagellate phenotype which results from the mutation, by substitution,addition and/or the deletion of bases or of a nucleotide fragment, ofthe nucleotide sequence according to any one of claims 1 to 3 of theflbA gene participating in the regulation of the biosynthesis of theflagellar proteins of H.pylori.
 13. Bacterial strain according to claim12, characterized in that it additionally lacks the hook protein ofH.pylori.
 14. Recombinant bacterial strain according to claim 12 orclaim 13, characterized in that it is obtained from the strain N6, whichwas deposited in the NCIMB on Jun. 26, 1992 under the number NCIMB40512.
 15. Recombinant bacterial strain according to either of claims 12and 14, characterized in that it is the strain N6flbA-, which wasdeposited in the NCIMB on Jun. 30, 1995 under the number NCIMB 40747.16. Bacterial strain according to any one of claims 12 to 15,characterized in that it is additionally mutated so that it produces anattenuated urease or else no longer produces urease, the mutationconsisting, for example, of a mutation of the nucleotide sequence of oneor more genes selected from among the genes ureA, ureB, ureC, ureD,ureE, ureF, ureG, ureH or ureI.
 17. Bacterial extract, characterized inthat it is an extract of bacterial strains according to any one ofclaims 12 to
 15. 18. Bacterial extract according to claim 17,characterized in that it is obtained after extracting with n-octylglucoside.
 19. Bacterial extract according to claims 17, characterizedin that it is obtained after extracting with PBS or with glycine. 20.Composition for the in vitro detection of an infection due to H.pyloriin a sample of biological fluid from a patient, in particular in asample of serum, which composition includes, as the active principle, abacterial strain according to any one of claims 12 to 15 or a bacterialextract according to any one of claims 17 to
 19. 21. Method for the invitro detection of an infection due to H.pylori in a sample ofbiological fluid from a patient, in particular in a sample of serum,which method comprises the steps of: bringing the sample under test intocontact with a bacterial strain according to any one of claims 12 to 15,or with a bacterial extract according to any one of claims 17 to 19,detecting an immunological reaction between the said bacterial strainand antibodies which are directed against H.pylori and which are presentin the sample under test.
 22. Immunogenic composition for obtainingantibodies against H.pylor, characterized in that it includes, as theactive principle, a bacterial strain according to any of claims 12 to 16or a bacterial extract according to any one of claims 17 to
 19. 23.Immunogenic composition for obtaining antibodies against H.pylor,characterized in that it includes an amino acid sequence according toeither of claims 10 and
 11. 24. Vaccinating composition for obtainingprotective antibodies against an infection due to H.pylori,characterized in that it includes, as the active principle, a bacterialstrain according to any one of claims 12 to 16 or a bacterial extractaccording to any one of claims 17 to
 19. 25. Vaccinating composition forobtaining antibodies against an infection due to H.pylori, characterizedin that it includes, as the active principle, antigens which are of theurnase type or which participate in the urease activity of H.pylori, inparticular antigens encoded by the genes ureA, ureB, ureC or ureD and aprotein having an amino acid sequence according to either of claims 10and
 11. 26. Monoclonal antibodies or polyclonal serum which is/aredirected against an amino acid sequence according to either of claims 10and
 11. 27. Monoclonal antibodies or polyclonal serum which is/aredirected against an H.pylori strain according to any one of claims 12 to15.
 28. Composition for the in vitro detection of an infection due toH.pylori in a biological sample, which composition includes, as theactive principle, monoclonal antibodies or a polyclonal serum whichis/are obtained against an H.pylori strain of the aflagellate phenotypeaccording to any one of claims 12 top
 15. 29. Use of the nucleotidesequences according to any of claims 1 to 9 for preparing immunogeniccompositions for obtaining antibodies against H.pylori.
 30. Kit fordiagnosing antibodies of patients infected with H.pylori, which kitincludes a bacterial extract according to any one of claims 15 to 19 andreagents which are required for demonstrating a reaction of theantigen/antibody type.